Thermodynamical interpretation of evolutionary dynamics on a fitness landscape in an evolution reactor,II

In our previous report [Aita, T., Morinaga, S., Hosimi, Y., 2004. Thermodynamical interpretation of evolutionary dynamics on a fitness landscape in an evolution reactor I. Bull. Math. Biol. 66, 1371–1403], an analogy between thermodynamics and adaptive walks on a Mt. Fuji-type fitness landscape in an artificial selection system was presented. Introducing the ‘free fitness’ as the sum of a fitness term and an entropy term and ‘evolutionary force’ as the gradient of free fitness on a fitness coordinate, we demonstrated that the adaptive walk (=evolution) is driven by the evolutionary force in the direction in which free fitness increases. In this report, we examine the effect of various modifications of the original model on the properties of the adaptive walk. The modifications were as follows: first, mutation distance d was distributed obeying binomial distribution; second, the selection process obeyed the natural selection protocol; third, ruggedness was introduced to the landscape according to the NK model; fourth, a noise was included in the fitness measurement. The effect of each modification was described in the same theoretical framework as the original model by introducing ‘effective’ quantities such as the effective mutation distance or the effective screening size.     

Adaptive walks by the fittest among finite random mutants on a Mt. Fuji-type fitness landscape II. Effect of small non-additivity

We examined properties of adaptive walks by the fittest on “rough Mt. Fuji-type” fitness landscapes, which are modeled by superposing small uncorrelated random component on an additive fitness landscape. A single adaptive walk is carried out by repetition of the evolution cycle composed of (1) mutagenesis process that produces random d-fold point mutants of population size N and (2) selection process that picks out the fittest mutant among them. To comprehend trajectories of the walkers, the fitness landscape is mapped into a (x, y, z)-space, where x, y and z represent, respectively, normalized Hamming distance from the peak on the additive fitness landscape, scaled additive fitness and scaled non-additive fitness. Thus a single adaptive walk is expressed as the dynamics of a particle in this space. We drew the “hill-climbing” vector field, where each vector represents the most probable step for a walker in a single step. Almost all of the walkers are expected to move along streams of vectors existing on a particular surface that overlies the (x, y)-plane, toward the neighborhood of a characteristic point at which a mutation-selection-random drift balance is reached. We could theoretically predict this reachable point in the case of random sampling search strategy. Received: 1 March 2000 / Published online: 3 August 2000     

Thermodynamical interpretation of an adaptive walk on a Mt. Fuji-type fitness landscape: Einstein relation-like formula holds in a stochastic evolution

We have theoretically studied the statistical properties of adaptive walks (or hill-climbing) on a Mt. Fuji-type fitness landscape in the multi-dimensional sequence space through mathematical analysis and computer simulation. The adaptive walk is characterized by the "mutation distance" d as the step-width of the walker and the "population size" N as the number of randomly generated d-fold point mutants to be screened. In addition to the fitness W, we introduced the following quantities analogous to thermodynamical concepts: "free fitness" G(W) is identical with W+T x S(W), where T is the "evolutionary temperature" T infinity square root of d/lnN and S(W) is the entropy as a function of W, and the "evolutionary force" X is identical with d(G(W)/T)/dW, that is caused by the mutation and selection pressure. It is known that a single adaptive walker rapidly climbs on the fitness landscape up to the stationary state where a "mutation-selection-random drift balance" is kept. In our interpretation, the walker tends to the maximal free fitness state, driven by the evolutionary force X. Our major findings are as follows: First, near the stationary point W*, the "climbing rate" J as the expected fitness change per generation is described by J approximately L x X with L approximately V/2, where V is the variance of fitness distribution on a local landscape. This simple relationship is analogous to the well-known Einstein relation in Brownian motion. Second, the "biological information gain" (DeltaG/T) through adaptive walk can be described by combining the Shannon's information gain (DeltaS) and the "fitness information gain" (DeltaW/T).     

Evolution of habitat selection: stochastic acquisition of cognitive clues?

Different habitat preferences in animals have been interpreted mostly as a result of different adaptive design of the species and/or as a result of interspecific competition. We propose an alternative view of evolution of habitat preferences. Our model is based on progressive stochastic acquisition of cognitive clues discriminating habitat features which correlate with expected fitness. We assume that acquisition of each cognitive clue allowing discrimination of ‘better’ and ‘worse’ habitats (according to the average fitness in each habitat) will constrain further evolution, because each further clue will discriminate habitats only within previously acquired preferences. Simple simulation model shows that if it is the case, even the species with equal habitat-related fitness differences will rapidly diversify in their habitat preferences. Therefore, similarly as in the evolution of other species-specific traits, the evolution of animal–habitat relationship may be strongly affected by stochastic events and historical contingency. This revised version was published online in July 2006 with corrections to the Cover Date.     

The expanded evolutionary synthesis—a response to Godfrey-Smith,Haig, and West-Eberhard

In responding to three reviews of Evolution in Four Dimensions (Jablonka and Lamb, 2005, MIT Press), we briefly consider the historical background to the present genecentred view of evolution, especially the way in which Weismann’s theories have influenced it, and discuss the origins of the notion of epigenetic inheritance. We reaffirm our belief that all types of hereditary information—genetic, epigenetic, behavioural and cultural—have contributed to evolutionary change, and outline recent evidence, mainly from epigenetic studies, that suggests that non-DNA heritable variations are not rare and can be quite stable. We describe ways in which such variations may have influenced evolution. The approach we take leads to broader definitions of terms such as ‘units of heredity’, ‘units of evolution’, and ‘units of selection’, and we maintain that ‘information’ can be a useful concept if it is defined in terms of its effects on the receiver. Although we agree that evolutionary theory is not undergoing a Kuhnian revolution, the incorporation of new data and ideas about hereditary variation, and about the role of development in generating it, is leading to a version of Darwinism that is very different from the gene-centred one that dominated evolutionary thinking in the second half of the twentieth century.     

A model for the emergence of adaptive subsystems

We investigate the interaction of learning and evolution in a changing environment. A stable learning capability is regarded as an emergent adaptive system evolved by natural selection of genetic variants. We consider the evolution of an asexual population. Each genotype can have ‘fixed’ and ‘flexible’ alleles. The former express themselves as synaptic connections that remain unchanged during ontogeny and the latter as synapses that can be adjusted through a learning algorithm. Evolution is modelled using genetic algorithms and the changing environment is represented by two optimal synaptic patterns that alternate a fixed number of times during the ‘life’ of the individuals. The amplitude of the change is related to the Hamming distance between the two optimal patterns and the rate of change to the frequency with which both exchange roles. This model is an extension of that of Hinton and Nowlan in which the fitness is given by a probabilistic measure of the Hamming distance to the optimum. We find that two types of evolutionary pathways are possible depending upon how difficult (costly) it is to cope with the changes of the environment. In one case the population loses the learning ability, and the individuals inherit fixed synapses that are optimal in only one of the environmental states. In the other case a flexible subsystem emerges that allows the individuals to adapt to the changes of the environment. The model helps us to understand how an adaptive subsystem can emerge as the result of the tradeoff between the exploitation of a congenital structure and the exploration of the adaptive capabilities practised by learning.     

Relative Fitness, Teleology, and the Adaptive Landscape

The metaphor of the adaptive landscape, introduced by Sewall Wright in 1932, has played, and continues to play, a central role in much evolutionary thought. I argue that the use of this metaphor is tied to a teleological view of the evolutionary process, in which natural selection directs evolution toward an improved future state. I argue further that the use of “relative fitnesses” standardized to an arbitrary value, which is closely connected with the metaphor of an adaptive landscape, produces a disconnect between the mean fitness of a population and any real property of that population. This allows for a vague and ill-defined improvement to occur under the influence of selection. Instead, I suggest that relative fitnesses should be standardized by the mean absolute fitness (expected population growth rate), so that they express the expected rate of increase in frequency, rather than number. Under this definition, the mean relative fitness of all populations is always 1.0, and never changes as long as the population continues to exist.     

Multiple mating in the ant Cataglyphis cursor: testing the sperm limitation and the diploid male load hypotheses

Multiple mating (i.e., polyandry) by queens in social Hymenoptera is expected to weaken social cohesion since it lowers within-colony relatedness, and hence, indirect fitness benefits from kin selection. Yet, there are many species where queens mate multiply. Several hypotheses have been put forward to explain the evolution and maintenance of polyandry. Here,we investigated the ‘sperm limitation’ and the ‘diploid male load’ hypotheses in the ant Cataglyphis cursor. Genetic analyses of mother-offspring combinations showed that queens mate with up to 8 males, with an effective mating frequency of 3.79. Significant paternity skew (unequal contribution of the fathers) was detected in 1 out of 5 colonies. The amount of sperm stored in the spermatheca was not correlated with the queen mating frequency, and males carry on average enough sperm in their seminal vesicles to fill one queen’s spermatheca. Analyses of the nuclear DNA-content of males also revealed that all were haploid. These results suggest that the ‘sperm limitation’ and the ‘diploid male load’ hypotheses are unlikely to account for the queen mating frequency reported in this ant. In light of our results and the life-history traits of C. cursor, we discuss alternative hypotheses to account for the adaptive significance of multiple mating by queens in this species. Received 13 August 2008; revised 19 November 2008; accepted 21 November 2008.     

Somatic embryogenesis from stigmas and styles of grapevine

Summary An in vitro protocol has been developed for callus indiction, somatic embryogenesis, and plant regeneration from stigma-style culture of grapevine. Four different grapevine cultivars (Vitis vinifera L.: cvs. ‘Bombino Nero’, ‘Greco di Tufo’, ‘Merlot’, and ‘Sangiovese’) were tested. Exlants were cultured on Nitsch and Nitsch medium (NN) supplemented with various combinations of 6-benzylaminopurine (BA: 4.5 and 9.0 μM) and β-naphthoxyacetic acid (NOA; 5.0 and 9.9 μM). Sucrose (88 mM) was used as the carbon source. Somatic embryogenesis was induced within 3–7 mo. after culture initiation. Even though explants of different origin (unfertilized ovules and anthers) regenerated somatic embryos, the higher embryogenic potential was observed in stigma and style explants, with the exception of ‘Merlot’, which regenerated somatic embryos only from unfertilized ovules. The percentages of stigma-style explants producing somatic embryos was 7% in ‘Bombino Nero’ (cultured on NN medium supplemented 9.0 μM BA and 9.9 μM NOA). 14% in ‘Greco di Tufo’ (4.5 μM BA and 9.9 μM NOA), and 8% in ‘Sangiovese’ (9.0 μM BA and 9.9 μM NOA). The presence of growth regulators (BA and NOA) in the medium was essential for induction of somatic embryogenesis. Plants were regenerated on hormone-free NN medium containing 88 mM sucrose.     

Molecular domestication – more than a sporadic episode in evolution

Transposable elements are short but complex pieces of DNA or RNA containing a streamlined minimal-genome with the capacity for its selfish replication in a foreign genomic environment. Cis-regulatory sections within the elements orchestrate tempo and mode of TE expression. Proteins encoded by TEs mainly direct their own propagation within the genome by recruitment of host-encoded factors. On the other hand, TE-encoded proteins harbor a very attractive repertoire of functional abilities for a cell. These proteins mediate excision, replication and integration of defined DNA fragments. Furthermore, some of these proteins are able to manipulate important host factors by altering their original function. Thus, if the host genome succeeds in domesticating such TE-encoded proteins by taming their ‘anarchistic behavior,’ such an event can be considered as an important evolutionary innovation for its own benefit. In fact, the domestication of TE-derived cis-regulatory modules and protein coding sections took place repeatedly in the course of genome evolution. We will present prominent cases that impressively demonstrate the beneficial impact of TEs on host biology over evolutionary time. Furthermore, we will propose that molecular domestication might be considered as a resumption of the same evolutionary process that drove the transition from ‘primitive genomes’ to ‘modern’ ones at the early dawn of life, that is, the adaptive integration of a short piece of autonomous DNA into a complex regulatory network. This revised version was published online in July 2006 with corrections to the Cover Date.     

Darwinian adaptation,population genetics and the streetcar theory of evolution

 This paper investigates the problem of how to conceive a robust theory of phenotypic adaptation in non-trivial models of evolutionary biology. A particular effort is made to develop a foundation of this theory in the context of n-locus population genetics. Therefore, the evolution of phenotypic traits is considered that are coded for by more than one gene. The potential for epistatic gene interactions is not a priori excluded. Furthermore, emphasis is laid on the intricacies of frequency-dependent selection. It is first discussed how strongly the scope for phenotypic adaptation is restricted by the complex nature of ‘reproduction mechanics’ in sexually reproducing diploid populations. This discussion shows that one can easily lose the traces of Darwinism in n-locus models of population genetics. In order to retrieve these traces, the outline of a new theory is given that I call ‘streetcar theory of evolution’. This theory is based on the same models that geneticists have used in order to demonstrate substantial problems with the ‘adaptationist programme’. However, these models are now analyzed differently by including thoughts about the evolutionary removal of genetic constraints. This requires consideration of a sufficiently wide range of potential mutant alleles and careful examination of what to consider as a stable state of the evolutionary process. A particular notion of stability is introduced in order to describe population states that are phenotypically stable against the effects of all mutant alleles that are to be expected in the long-run. Surprisingly, a long-term stable state can be characterized at the phenotypic level as a fitness maximum, a Nash equilibrium or an ESS. The paper presents these mathematical results and discusses – at unusual length for a mathematical journal – their fundamental role in our current understanding of evolution. Received 22 April 1994; received in revised form 10 July 1995     

Darwin without borders? Looking at ‘generalised Darwinism’ through the prism of the ‘hourglass model’

This article critically analyzes the arguments of the ‘generalized Darwinism’ recently proposed for the analysis of social-economical systems. We argue that ‘generalized Darwinism’ is both restrictive and empty. It is restrictive because it excludes alternative (non-selectionist) evolutionary mechanisms such as orthogenesis, saltationism and mutationism without any examination of their suitability for modeling socio-economic processes and ignoring their important roles in the development of contemporary evolutionary theory. It is empty, because it reduces Darwinism to an abstract triple-principle scheme (variation, selection and inheritance) thus ignoring the actual structure of Darwinism as a complex and dynamic theoretical structure inseparable from a very detailed system of theoretical constraints. Arguing against ‘generalised Darwinism’ we present our vision of the history of evolutionary biology with the help of the ‘hourglass model’ reflecting the internal dynamic of competing theories of evolution.     

Efficient and simple plant regeneration via organogenesis from leaf segment cultures of persimmon (Diospyros kaki thunb.)

Summary An efficient and simple plant regeneration system via organogenesis from leaf segments of persimmon (Diospyros kaki Thunb.) cultivars ‘Fuyu’ and ‘Nishimurawase’ has been developed. The regeneration capacity was influenced by the culture vessels, gelling agents, plant growth regulators, and light conditions. Leaf explants taken from in vitro shoots were cultured on a modified Murashige and Skoog medium (MS1/2N), for 16 wk without transfer to fresh medium. Adventious shoots appeared after 4 and 8 wk in culture of ‘Nishimurawase’ and ‘Fuyu’ tissues, respectively. The culture of leaf explants in Erlenmeyer flasks with medium containing 4 g l⁻¹ agar enhanced shoot formation in comparison to media with increased agar concentrations. Optimal shoot regeneration was obtained with 5 mg l⁻¹ (22.8 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) indole-3-butyric acid (IBA) for ‘Nishimurawase’, and 10 mg l⁻¹ (45.6 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) IBA for ‘Fuyn’. Shoot regeneration frequencies in both cultivars were 100%, and shoot numbers per explant reached up to 9.2 for ‘Nishimurawase’ and 2.2 for ‘Fuyu’. Dark incubation during the first 4–5 wk was the most effective condition to successfully influence shoot regeneration in both cultivars. While dark incubation was essential for adventitious shoot formation by ‘Fuyu’, it was only slightly beneficial to ‘Nishimurawase’. More than 80% of the regenerated shoots rooted within 4 wk on hormone-free MS1/2N demium after having been dipped for 30 s in 250 mg l⁻¹ (1.1. mM) IBA solution.     

Fitness landscape of sympatric pupfishes a useful tool for visualizing speciation

Arguably the most useful model of evolution emerged from the mind of Sewall Wright when he invented the fitness landscape (Wright 1932 ). In a recent issue of Molecular Ecology, Martin & Feinstein ( 2014 ) investigate the genetics and demographic history of an adaptive radiation of pupfish on San Salvador Island. Since the founder species colonized the island 10 000 years ago, two descendent species have appeared and in several lakes all three species (a durophage, a scale‐eater, and the generalist ancestral form) coexist. The three species are thought to occupy three distinct fitness peaks. The durophage and generalists' peaks are close, whereas the scale‐eater's peak is predicted to be distant and separated from the other two by a deep valley. Consistent with this view, gene flow between the two species on close fitness peaks is greater than the gene flow between these two species and the third species on a more distant peak. Correspondingly, the inferred fitness landscape predicts progress towards speciation, with more limited separation of species on close peaks, and that speciation is more complete for the scale‐eater. The article provides an illustrative example of the power afforded by analysis of large numbers of SNPs for estimating key parameters underlying evolutionary divergence.     

MHC-associated mating strategies and the importance of overall genetic diversity in an obligate pair-living primate

Mate choice is one of the most important evolutionary mechanisms. Females can improve their fitness by selectively mating with certain males. We studied possible genetic benefits in the obligate pair-living fat-tailed dwarf lemur (Cheirogaleus medius) which maintains life-long pair bonds but has an extremely high rate of extra-pair paternity. Possible mechanisms of female mate choice were investigated by analyzing overall genetic variability (neutral microsatellite marker) as well as a marker of adaptive significance (major histocompatibility complex, MHC-DRB exon 2). As in human medical studies, MHC-alleles were grouped to MHC-supertypes based on similarities in their functional important antigen binding sites. The study indicated that females preferred males both as social and as genetic fathers for their offspring having a higher number of MHC-alleles and MHC-supertypes, a lower overlap with female’s MHC-supertypes as well as a higher genome wide heterozygosity than randomly assigned males. Mutual relatedness had no influence on mate choice. Females engaged in extra-pair mating shared a significant higher number of MHC-supertypes with their social partner than faithful females. As no genetic differences between extra-pair young (EPY) and intra-pair young (IPY) were found, females might engage in extra-pair mating to ‘correct’ for genetic incompatibility. Thus, we found evidence that mate choice is predicted in the first place by the ‘good-genes-as-heterozygosity hypothesis’ whereas the occurrence of extra-pair matings supports the ‘dissassortative mating hypothesis’. To the best of our knowledge this study represents the first investigation of the potential roles of MHC-genes and overall genetic diversity in mate choice and extra-pair partner selection in a natural, free-living population of non-human primates.     

Necessity,Futility and the Possibility of Defining Life are all Embedded in its Origin as a Punctuated-gradualism

The criteria used for defining life are influenced by various philosophical visions about life, ranging from holism to reductionism and from mechanistic-reductionism to vitalism. Using different scenarios about the origin and evolution of life as well as properties of energy-dissipative systems, artificial life simulations and basic tenets of xenobiology, guidelines can be established for formulating a definition of life. A definition of life is proposed that is parametric, non-Earth-centric, quantitative and capable of discriminating ‘living entities’ from ‘life’. Living entities are defined as self-maintained systems, capable of adaptive evolution individually, collectively or as a line of descend. Life is a broader concept indicating that the capacity to express these attributes is either virtual or actual. At least four major phase transitions can be recognized during the origin of life (reflexive activity; self-regulated homeostasis; the advent of informatons and the origin of adaptive evolution); these make the origin and evolution of early life an example of ‘punctuated gradualism’. Such phase transitions can be used to identify a boundary in early evolution where life began. This contribution identifies the step in the evolution of a dynamic system when digital control of the system’s state becomes dominant over analogical control, and genetic information is irreversibly used for adaptive evolution, as the boundary between non-living and living systems.     

Chlorophyll content and leaf elongation rate in wheat seedlings as a measure of manganese tolerance

After aluminum toxicity, manganese (Mn) toxicity is probably the second most important growth limiting factor in acid soils. The purpose of this study was to determine the feasibility of using chlorophyll content and leaf elongation rate (LER) for regrowth of Mn stressed seedlings as a rapid seedling based screening bioassay for Mn tolerance in segregating populations of wheat (Triticum aestivum L.). In one experiment, chlorophyll was determined for the cultivars Norquay (Mn-tolerant) and Columbus (Mn-sensitive) subjected to twelve Mn levels (2 to 2000 μM) in nutrient solutions. As Mn concentration increased, chlorophyll ‘a’ and ‘b’ contents of the Mn-tolerant cultivar decreased up to 9%, while in the Mn-sensitive cultivar it was reduced by as much as 43%. The chlorophyll ‘a/b’ ratio did not differ among Mn concentrations for either cultivar. In a second experiment, chlorophyll content and LER for regrowth of Mn stressed seedlings (1000 μM) was determined for Columbus and Katepwa (Mn-sensitive), Oslo (Mn-intermediate), and Norquay and Laura (Mn-tolerant). Manganese tolerance as assayed by chlorophyll ‘a’ and ‘b’ and LER was significantly correlated with Mn tolerance as assayed by the relative root weight methodology (RRW). Thus, chlorophyll content of Mn-stressed seedlings and LER of seedling regrowth appear to be suitable techniques for screening unreplicated selections of segregating populations for tolerance to Mn.     

Sources for Imanishi Kinji’s views of sociality and evolutionary outcomes

Prior to the contribution of genetics or the modern evolutionary synthesis (MES) to natural selection theory, social ecologists searched for factors in addition to natural selection that could influence species change. The idea that sociality, not just biology, was important in determining evolutionary outcomes was prevalent in research in social ecology in the 1920s and 1930s. The influence of ‘tradition’ (or the transmission of learned behaviours between generations) and the view that animals are active in selecting their own environments, rather than passive organisms acted upon by chance, were given as much attention as natural selection theory in European ecology, while animal aggregation and cooperation studies were pursued in America. Imanishi Kinji’s personal library and his scientific notes and papers reveal that he was well aware of this literature and had been profoundly influenced by these earlier viewpoints prior to writing his view of nature in his first book, Seibutsu no Sekai (The World of Living Things, 1941). Evidence is presented to show that he developed his theories based partly on early western debates in social ecology while finding inspiration and a way to express his views in the writings of philosopher Nishida Kitarō and, perhaps, General J C Smuts. One of Imanishi’s lasting contributions is in the demonstrated results of over 40 years of subsequent ecological and ethological research by Imanishi and those trained by him that maintained the broader viewpoints on evolution that had been dropped from the western corpus of research by the 1950s. The current attempt to again get beyond natural selection theory is reflected in debates surrounding genetic and cultural evolution of cooperation, the biology of ‘traditions’ and the idea of ‘culture’ in animal societies. Imanishi Kinji is the Japanese name order, with family name first. Other Japanese names in the text are also written with family name first. A modified version of this paper appeared in Japanese in Seibutsu Kagaku, Vol. 57 No. 3, April 2006, pp 142–149.     

Long-term study of somatic embryogenesis from anthers and ovaries of 12 grapevine (<Emphasis Type="Italic">Vitis</Emphasis> sp.) genotypes

Summary Anthers and ovaries of six grapevine cultivars (three Vitis vinifera L., two V × Labruscana L. H. Bailey, and one complex hybrid) were extracted from flower buds over 2 yr and cultured on three media reported to promote somatic embryogenesis in Vitis tissues. The highest percent embryogenesis from the hybrid ‘Chancellor’ and V. vinifera ‘Chardonnay’, ‘Merlot’, and ‘Pinot Noir’ occurred on medium C [Nitsch and Nitsch, 1969, basal medium with 3.0% (w/v) sucrose, 0.01% (w/v) inositol. 0.3% (w/v) Phytagel, 2.5 μM 2.4-dichlorophenoxyacetic acid, 2.5μM β-naphthoxyacetic acid, 5.0μM N-(2-chloro-4-pyridyl)-N′-phenylurea, and 0.05% (w/v) glutamine]. Regardless of the media, the labrusca cultivars ‘Concord’ and ‘Niagara’ produced soft non-embryogenic callus that was sometimes mixed with well-developed somatic embryos. Nine vinifera genotypes were further tested for several different years on medium C. Embryogenic cultures suitable for transformation were obtained from all genotypes in more than 1 yr. The average percent embryogenesis from ovaries was 7-fold higher than from anthers. There was significant annual variation in percent embryogenesis, demonstrating the need for media comparisons to be replicated for more than one season. Suspension cultures suitable for use in genetic transformation were initiated from ‘Chardonnay’, ‘Merlot,’ and ‘Pinot Noir’ pro-embryogenic masses. ‘Chardonnay’ suspension cultures plated and grown under conditions developed for recovery of plants after biolistic transformation yielded approximately 500 non-transformed embryos per plate after 4 mo. of culture, with 68.6% of the embryos converting to plants. This is the first reported protocol for embryogenesis from ‘Concord,’ ‘Cabernet Franc,’ and ‘Pinot Noir’ grapevines.